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Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Delta2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.

Certain proteins can undergo polyglycylation and polyglutamylation. Polyglutamylases (glutamate ligases) have recently been identified in a family of tubulin tyrosine ligase-like (TTLL) proteins. However, no polyglycylase (glycine ligase) has yet been reported. Here we identify a polyglycylase in the TTLL proteins by using an anti-poly-glycine antibody. The antibody reacted with a cytoplasmic 60-kDa protein that accumulated in elongating spermatids. Using tandem mass spectrometry of trypsinized samples, immunoprecipitated by the antibody from the TTLL10-expressing cells, we identified the 60-kDa protein as nucleosome assembly protein 1 (NAP1). Recombinant TTLL10 incorporated glycine into recombinant NAP1 in vitro. Mutational analyses indicated that Glu residues at 359 and 360 in the C-terminal part of NAP1 are putative sites for the modification. Thus, TTLL10 is a polyglycylase for NAP1.

Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.

The alpha- and beta-tubulins present in cytoskeletons of Tritrichomonas mobilensis are extensively glutamylated. Automated sequencing and mass spectrometry of the carboxyterminal peptides identifies 4 glutamylation sites in alpha- and 2 sites in beta-tubulin. They are marked by asterisks in the terminal sequences GDE*E*E*E*DDG (alpha) and EGE*E*DEEAEA (beta). This is the first report that tubulin glutamylation can occur at multiple sites. Although T. mobilensis has four flagellae the tubulins lack polyglycylation. Thus glycylation is not necessary for formation or function of axonemal microtubules. Alpha-tubulin is completely acetylated at lysine 40 and shows no tyrosine cycle. Peptide sequences establish two distinct beta-tubulins.

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis.

We have examined the distribution of glutamylated tubulin in non-neuronal cell lines. A major part of centriole tubulin is highly modified on both the alpha- and beta-tubulin subunits, whereas a minor part of the cytoplasmic tubulin is slightly modified, on the beta-tubulin only. Furthermore, we observed that tubulin glutamylation varies during the cell cycle: an increase occurs during mitosis on both centriole and spindle microtubules. In the spindle, this increase appears more obvious on the pole-to-pole and kinetochore microtubules than on the astral microtubules. The cellular pattern and the temporal variation of this post-translational modification contrast with other previously described tubulin modifications. The functional significance of this distribution is discussed.

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement

This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosination/tyrosination, acetylation/deacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable alpha-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and alpha-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulin/microtubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications.

We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia , which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of α-tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine-tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin-specific polyglycylation could be as old as tubulin and axonemal structures.

Microtubular networks are extensively developed in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Edde et al., 1990: Science 247:82-85; Edde et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules. Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies.

Limited subtilisin digestion of the tubulin alpha, beta heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both alpha and beta subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of alpha and Gln-433 and His-396 of beta tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (alpha) and His-396 (beta) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or -435 of alpha and beta tubulin, respectively.

We describe the presence of alpha-tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly-disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both alpha-tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu-C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained epsilon N-acetyllysine at position 40 of the alpha-tubulin molecule. This result demonstrates that mouse brain alpha-tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated alpha-tubulin was resolved into five isoelectric variants, denoted alpha 3 and alpha 5 to alpha 8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of alpha-tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal alpha-tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.

The tubulin molecule consists of an alpha- and a beta-subunit, each of which exists in several isotypic forms. It has been previously shown that one of the isotypes of neuroblastoma beta-tubulin is phosphorylated at a serine residue in vivo [(1985) J. Cell Biol. 100, 764-774]. Here we identify the phosphorylated isotype as beta 2 (type III). Moreover, the large size of the phosphorylated tryptic peptide and sequence comparisons of vertebrate beta-tubulins suggest that one of the two serines in positions 444 and 446 is the phosphorylated residue. Our results raise the possibility that beta 2-tubulin differs functionally from the other beta-tubulin isotypes.